Labelled adrenomedullin derivatives and their use for imaging and therapy

ABSTRACT

The present invention relates to an adrenomedullin derivative including an adrenomedullin peptide, or a fragment thereof chelated or otherwise bound to at least one active agent. Examples of active agents include a paramagnetic element, a radioactive element and a fibrinolytic agent, among others. Paramagnetic agents have a distribution that is relatively easily shown through Magnetic Resonance Imaging (MRI). Radioactive agents have applications in imaging and delivery of radiations, depending on the specific element included in the active agent. Delivery of fibrinolytic agents mainly to a specific organ, such as for example to the lungs, allows to substantially improve the specificity and efficacy of thrombolytic therapy by allowing local delivery of the fibrinolytic agent, thereby reducing the risks of major bleeding in the therapy of the organ. If the organ is the lungs, a non-limiting example of pathology treatable with the fibrinolytic agent is pulmonary embolus.

This application claims priority from U.S. Provisional PatentApplication Ser. No. 60/924,393 filed May 11, 2007. This application isalso a continuation-in-part of PCT Application PCT/CA2005/000791 filedMay 24, 2005, which entered National Phase in the United States, whichclaimed priority from U.S. Provisional Patent Application Ser. No.60/573,334 filed May 24, 2004.

FIELD OF THE INVENTION

The present invention relates to the use of labelled compounds forimaging or therapy. More specifically, the present invention isconcerned with labelled adrenomedullin derivatives and their use forimaging and therapy.

BACKGROUND OF THE INVENTION

A currently existing agent for the clinical imaging of pulmonarycirculation is ^(99m)Tc-labelled albumin macroaggregates. This agent isused for the diagnosis of physical defects of the circulation due topulmonary embolus. This agent is larger than small pulmonary vessels.Accordingly, further to being injected in an animal, this agent istrapped by these small pulmonary vessels, which enables externaldetection.

Important limitations of albumin macroaggregates include the inabilityto image the small pulmonary circulation beyond the point ofobstruction. This limits the sensitivity of this substance to detectsmall vascular defects. Also, there are potential infectious risks sincealbumin macroaggregates are derived from human albumin. Additionally,albumin macroaggregates are unable to detect functional (biological)defects of the pulmonary circulation since their retention is uniquelydependent on physical characteristics of the vessels.

In addition, many other pulmonary diseases are difficult to diagnose.For example, pulmonary arterial hypertension (PAH) is a disordercharacterized by endothelial dysfunction with intimal as well asvascular smooth muscle proliferation leading to gradual obliteration ofpulmonary arterioles [36]. Screening for PAH is performed bytransthoracic Doppler echocardiography with estimation of the pulmonaryartery systolic pressure using the tricuspid valve regurgitant jet.Although this approach correlates with hemodynamically measuredpulmonary pressure, it does not provide direct information on thebiology of the pulmonary circulation and may miss the early presence ofpulmonary vascular disease. The recent availability of oral therapiesfor PAH such as endothelin receptor antagonists and phophodiesteraseinhibitors advocate for earlier diagnosis of this condition andtreatment of subjects in functional class II. There is thereforeimperious necessity for novel diagnostic approaches of this appallingcondition that could provide earlier and more precise diagnosis.

There exist compounds that have an affinity for particular organs, suchas for example adrenomedullin (AM). AM is a 52-amino-acidmultifunctional regulatory peptide highly expressed in endothelial cellsand widely distributed in various tissues [1,2]. The structure of AM iswell conserved across species, with only six substitutions and twodeletions in the rat [rAM(1-50)] compared with the human [hAM(1-52)][3]. AM possesses structural homology with CGRP (calcitonin gene-relatedpeptide), making it a member of the calcitonin/CGRP/amylin family(CT/CGRP/AMY peptide family).

The biological activities of AM are mediated by receptors composed oftwo essential structural components: a seven-transmembrane protein, thecalcitonin receptor-like receptor (CRLR), and a single transmembranedomain termed RAMP (receptor-activity-modifying protein) [4,5]. Theassociation of CRLR/RAMP1 represents the CGRP1 receptor and is notspecific to AM. At the opposite, a specific AM receptor comes from thecoupling of CRLR/RAMP2 or CLRL/RAMP3 [6]. This specific AM receptor canbe blocked by the C-terminal AM fragment [hAM(22-52)][7].

A biological action of AM is a potent hypotensive effect. The systemichypotensive action of AM can however be reduced and sometimes abolishedafter intravenous compared with intra-arterial infusion [8], suggestingthat the lungs have a potential to clear circulating AM and modulate itscirculating levels. Many studies have confirmed that AM is cleared bythe pulmonary circulation [9-12]. However, the relative contribution ofthe lungs to AM clearance in comparison with other organs has not beensystematically evaluated and, more specifically, single-pass pulmonaryclearance of AM has not been quantified in vivo. In addition, the exactregion of the AM peptide that is responsible for the hypotensive effectsis currently unknown.

Against this background, there exists a need in the industry to providenovel compounds having an affinity for the lungs, the kidneys and otherorgans, and more specifically to provide such compounds suitable for usein therapy and imaging.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

OBJECTS OF THE INVENTION

An object of the present invention is therefore to provide novelcompounds having an affinity for the lungs.

SUMMARY OF THE INVENTION

In a first broad aspect, the invention provides an adrenomedullinderivative comprising: an adrenomedullin peptide or a fragment thereofbound with at least one active agent, the adrenomedullin peptide being amammalian adrenomedullin peptide. In some embodiments, the active agentcomprises a radioactive element. In other embodiments, the active agentincludes any other suitable detectable label.

Typically, the adrenomedullin peptide comprises a peptide having thesequence:Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr(SEQ ID NO:1) or a fragment thereof and is in a linear form or in acyclic form.

In some embodiments, the adrenomedullin peptide comprises anadrenomedullin fragment having the sequence:Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr(SEQ ID NO:2) or a fragment thereof

In other embodiments, the adrenomedullin peptide comprises anadrenomedullin fragment having the sequence:X1-X2-X3-X4-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr(SEQ ID NO:3), wherein: X1 is absent or is selected from the groupconsisting of:Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly (SEQ IDNO:4), Ser-Phe-Gly (SEQ ID NO:5) and Gly-Gly-Ala-Gly (SEQ ID NO:6); X2is Cys or HomoCys; X3 is Arg-Phe-Gly-Thr (SEQ ID NO:7) or a linear orbranched PEG moiety having at least two Poly(ethylene glycol) (PEG)subunits; and X4 is Cys or HomoCys. In some examples, X3 is a linear PEGmoiety having 2 or 4 PEG subunits. Advantageously, such PEG moietieshave linear dimensions substantially similar to linear dimensions of anamino acid sequence that X3 replaces when compared to human AM. It ishypothesized that in these embodiments, the proposes AM has goodbiological properties as the structure of the AM is not modified greatlywhen compared to AM in which X3 is a peptide.

In a specific embodiment of the invention, X1 is Gly-Gly-dAla-Gly (SEQID NO:8). In other specific embodiments, X1 is Gly-Gly-Ala-Gly (SEQ IDNO: 6), X2 is Cys, X3 is a linear moiety having 2 or 4 PEG subunits, andX4 is Cys.

In some examples, wherein the radioactive element is bound directly toan amino acid of the adrenomedullin peptide. Examples of radioactiveelement include: ^(99m)Tc, ⁶⁷Ga, ⁶⁴Cu, ⁹⁰Y, ¹⁶¹Tb, ¹⁷⁷Lu, and ¹¹¹In.

In some examples, the adrenomedullin peptide is in a linear form and theradioactive element is bound directly to a cysteine amino acid of theadrenomedullin peptide.

In other embodiments of the invention, the active agent comprises aparamagnetic element. In yet other embodiments of the invention, theactive agent is an element selected from the group consisting of: Fe,Ca, Mn, Mg, Cu, and Zn. In yet other embodiments of the invention, theactive agent is selected from the group consisting of: active agentscomprising at least one paramagnetic element, active agents comprisingat least one radioactive element, and fibrinolytic agents.

In another broad aspect, the invention provides a method of determininga disease state in an organ in a mammal, the organ comprisingadrenomedullin-receptor-bearing cells, the method comprising: a)administering to the mammal a labelled adrenomedullin derivative in aneffective amount to achieve binding between the labelled adrenomedullinderivative and the adrenomedullin-receptor-bearing cells; b) generatingan image of the distribution of the labelled adrenomedullin derivativein the organ of the mammal; c) using the image of step b) to determine alabelling pattern of the adrenomedullin derivative in the organ; d)comparing the labelling pattern of step c) to a labelling pattern of anon diseased organ; and e) determining the disease state of the organ atleast in part on the comparison of step d).

For example, the organ is a lung. In this example, in a specificexample, the disease is pulmonary embolus and wherein the labellingpattern of step c) indicates that the labelled adrenomedullin derivativeis present in a greater concentration in upstream regions of the lungthan in downstream regions of the lung. Upstream and downstream regionsare defined with respect to blood flow in the lungs. Detecting thepulmonary embolus may include identifying a labelling pattern in aregion of the lung in which the labelled adrenomedullin derivative ispresent in a lower concentration than in adjacent regions of the lung.In another specific example, the disease is pulmonary arterialhypertension. And detecting the pulmonary arterial hypertension includesdetecting a reduced uptake of the labelled adrenomedullin derivative ascompared to a baseline uptake of the labelled adrenomedullin derivativein the non diseased organ, wherein the reduced uptake indicatespulmonary arterial hypertension.

In another example, the organ is a kidney and the disease is kidneydamage. Determining the kidney damage may include identifying alabelling pattern in a region of the kidney in which the labelledadrenomedullin derivative is present in a lower concentration than inadjacent regions of the kidney. Determining the kidney damage may alsoinclude detecting a reduced uptake of the labelled adrenomedullinderivative as compared to a baseline uptake of the labelledadrenomedullin derivative in the non diseased organ, wherein the reduceduptake indicates kidney damage.

In some embodiments, the proposed method has one or more of thefollowing features: the mammal is human; the labelled adrenomedulinderivative is administered to the mammal at a substantiallyhemodynamically inactive dose; the labelled adrenomedullin derivative islabelled with a radioactive element and the labelled adrenomedullinderivative is one of the labelled adrenomedullin derivatives describedhereinabove.

In another broad aspect, the invention provides a method of determiningthe presence and density of adrenomedullin receptor-bearing cells in amammal comprising: administering to the mammal an effective amount oflabelled adrenomedullin derivative to achieve binding between thelabelled adrenomedullin derivative and adrenomedullin-receptor-bearingcells; and determining the distribution of the labelled adrenomedullinderivative to obtain an image of the adrenomedullin-receptor-bearingcells.

In another broad aspect, the invention provides the use of labelledadrenomedullin derivatives to image the lungs or the kidneys of amammal.

In another broad aspect, the invention provides, the invention providesan adrenomedullin derivative comprising: an adrenomedullin peptide, theadrenomedullin peptide comprising a peptide having the sequence:Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr(SEQ ID NO:1) or a fragment thereof, the adrenomedullin peptide being amammalian adrenomedullin peptide. Examples of such adrenomedullinpeptides are found hereinabove. In some embodiments the adrenomedullinderivative further comprises a chelating peptide covalently bound to theadrenomedullin peptide and at least one active agent bound to thechelating peptide.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of preferred embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIG. 1 illustrates the plasma kinetics of ¹²⁵I-rAM(1-50) after a singleintravenous injection in a rat;

FIG. 2 illustrates the biodistribution of ¹²⁵I-rAM(1-50) afterintravenous injection in rats (†P<0.001 compared with the lungs;*P<0.005 and § P<0.001 compared with control, n=10/group);

FIG. 3 illustrates the pulmonary retention of ¹²⁵I-rAM(1-50) in ratsafter intravenous and intra-arterial injection (\P<0.005 and § P<0.001compared with venous injection, n=10/group);

FIG. 4 illustrates an example of indicator-dilution curve outflowprofiles in the canine pulmonary circulation in control conditions (A)and after injection of unlabelled rAM(1-50) (B), Insets showing thenatural log ratio curves of the tracers (FR is fractional recovery ofeach tracer);

FIG. 5 illustrates the plasma kinetics of ^(99m)Tc-DTPA-hAM1-52 aftersingle intravenous injections in dogs, the data being fitted with atwo-phase exponential decay equation (the inset shows a logarithmicscale, n=6/group);

FIG. 6 illustrates the biodistribution of ^(99m)Tc-DTPA-hAM1-52 afterintravenous injection in dogs (†P<0.005 vs. 30 minutes; *P<0.001 vs. 30minutes; §P<0.001 vs. lungs, n=6/group);

FIG. 7 illustrates the dynamic biodistribution of ^(99m)Tc-DTPA-hAM1-52after intravenous injection in dogs (n=6/group);

FIG. 8 is a gamma camera image of a dog's thorax obtained further to aninjection of ^(99m)TC marked AM; and

FIG. 9 illustrates selective surgical pulmonary lobectomy effects on^(99m)Tc-DTPA-hAM1-52 perfusion in dog as imaged through a gamma camera:(A) anterior view, (B) oblique view, a wedge shaped perfusion defectbeing indicated by an arrow and delimitated by dotted lines.

FIG. 10, in X-Y graphs, illustrates the time-dependent biodistributionin various organs of ^(99m)Tc-linear AM after intravenous injection indogs;

FIG. 11 in a X-Y graph, illustrates the plasma kinetics of^(99m)Tc-linear AM after a single intravenous injection in rats;

FIG. 12, in a bar chart, illustrates the biodistribution in variousorgans of ^(99m)Tc-linear AM 30 minutes after intravenous injection inrats;

FIG. 13, in a bar chart, illustrates the biodistribution in variousorgans of ^(99m)Tc-linear AM 30 and 60 minutes after intravenousinjection in rats;

FIG. 14, in a bar chart, illustrates the purification of ^(99m)Tc-linearAM using Sep Pak cartridges. The labelled compound is separated fromcolloids and free ^(99m)Tc by instant thin layer chromatography (ITLC).After a first and second pass on a column, purified fractions of 91% and94% respectively are obtained. This level of purification is suitablefor in vivo utilization;

FIG. 15, in a bar chart, illustrates the stability of purified^(99m)Tc-linear AM at room temperature over periods of 24 and 48 hoursas assessed by instant thin layer chromatography (ITLC). In a solutionof phosphate buffered saline (PBS) the purified compound is stable andmaintains radiochemical purity of 94% for up to 48 hours;

FIG. 16, in a X-Y graph, illustrates an example of a first passpulmonary clearance experiment of ^(99m)Tc-linear AM in a dog in vivo. Abolus containing trace amounts of ^(99m)Tc-linear AM (TC99) and Evansblue dye labelled albumin (EBD) are injected in the pulmonary artery andtimed outflow samples are collected over 35 seconds. The concentrationin each sample is plotted as a function of time to construct andindicator-dilution curve. The differential area between the curvesrepresents mean tracer ^(99m)Tc-linear AM extraction within a singlepulmonary passage. In this example, mean extraction was 21%;

FIGS. 17A, 17B and 17C, in X-Y graphs, illustrate the displacement of^(99m)TC marked cyclic AM by various ^(99m)TC marked AM derivatives as afunction of concentration of the ^(99m)TC marked AM variantstime-dependent biodistribution;

FIG. 18 is a gamma camera image of a dog's thorax obtained further to aninjection of ^(99m)TC marked linear AM showing a substantiallyhomogeneous lung uptake of the tracer, thereby enabling externalimaging;

FIG. 19 is a gamma camera image of a mouse's thorax and abdomen obtainedfurther to an injection of ^(99m)TC marked linear AM showing asubstantially homogeneous lung uptake of the tracer, thereby enablingexternal imaging.

FIG. 20, in a bar chart, illustrates the biodistribution in variousorgans of ^(99m)Tc-cyclic AM and ^(99m)Tc-linear AM 30 min and 60 minafter intravenous injection in dogs;

FIG. 21, in a X-Y graph, illustrates the plasma kinetics of^(99m)Tc-cyclic hAM and ^(99m)Tc-linear human AM (hAM) after a singleintravenous injection in dogs;

FIG. 22 is a whole body gamma camera image of a dog obtained further toan injection of ^(99m)TC marked linear AM. There is homogeneous kidneyuptake of the tracer enabling external imaging.

FIG. 23 represent tomographic gamma camera images of dog kidneysobtained further to an injection of ^(99m)TC marked linear AM intransverse, sagittal and coronal sections. There is homogeneous kidneyuptake of the tracer that is limited to the kidney cortex enablingexternal imaging.

FIG. 24 represents kidney and bladder uptake of ^(99m)TC marked linearAM further to injection to control rats and to rats with kidney damageinduces by monocrotaline injection (MCT). There is more than 50%reduction in relative kidney uptake after MCT suggesting that this agentcould be used in the diagnosis of kidney disorders.

FIG. 25 illustrates the plasma kinetics of ^(99m)TC marked linear AM ina control group and in a monocrotaline-induced pulmonary arterialhypertension (PAH) (MCT) group. The fitted two-compartment model curvesare significantly different with p<0.001.

FIG. 26 illustrates in vivo biodistribution of ^(99m)TC marked linear AMin the control group and in the monocrotaline-induced PAH (MCT) group. #p<0.001 versus control, *p<0.05 versus control.

FIG. 27 illustrates ex vivo biodistribution of ^(99m)TC marked linear AMin the control group and in the monocrotaline-induced PAH (MCT) group.#p<0.001 versus control, *p<0.05 versus control.

FIG. 28 illustrates whole body scans 30 minutes following intravenous^(99m)Tc-AM-L injection. A) control animal B) PAH model animal.

FIG. 29 illustrates lung tissue protein expression of the AM receptorcomponent RAMP2 in Sham and MCT treated rats;

FIG. 30 illustrates in table form various AM derivatives that weretested for biological activity;

FIG. 31 illustrates the biodistribution of various AM derivativesradiolabelled with ^(99m)Tc. The experiments were realized in vivo indogs and the activity of each organ determined 30 minutes afterinjection using a gamma camera;

FIG. 32 illustrates the lung kinetics of AM derivatives in the first 120minutes following injection in dogs. Of note is the plateau displayed bythe AM derivative identified as DFH-12 in the table of FIG. 30 between30 seconds and 45 minutes following injection; and

FIG. 33 compares images obtained with two different adrenomedullinderivatives (identified by DFH-08 and DFH-12 in the table of FIG. 30) 30minutes after injection in rats. Both tracers display detectable lungsand kidneys uptake. The DFH-12 however does not display as much liveractivity enabling better delineation of the lungs.

DETAILED DESCRIPTION

The present invention relates to the use of an adrenomedullin derivativeincluding an adrenomedullin peptide chelated to, or otherwise bound to,at least one active agent. For example, the adrenomedullin peptidecomprises adrenomedullin having the sequence:Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr(SEQ ID NO:1), or a fragment thereof. This adrenomedullin peptidecorresponds to amino acids 1-52 of human adrenomedullin. Additionally,in other embodiments of the invention, fragments of adrenomedullincorrespond to shorter peptide sequences, such as amino acids 1-50 of ratadrenomedullin or any other suitable fragment of any mammalianadrenomedullin.

Examples of active agents include a paramagnetic element, a radioactiveelement and a fibrinolytic agent, among others. Paramagnetic agents havea distribution that is relatively easily shown through MagneticResonance Imaging (MRI). Radioactive agents have applications in imagingand delivery of radiations, depending on the specific element includedin the active agent. Delivery of fibrinolytic agents mainly to aspecific organ, such as for example to the lungs, allows tosubstantially improve the specificity and efficacy of thrombolytictherapy by allowing local delivery of the fibrinolytic agent, therebyreducing the risks of major bleeding in the therapy of the organ. If theorgan is the lungs, a non-limiting example of pathology treatable withthe fibrinolytic is pulmonary embolus.

Non-limiting examples of radioactive elements suitable for imaginginclude: ^(99m)Tc, ¹¹¹In, ⁶⁷Ga, ⁶⁴Cu, ⁹⁰Y, ¹⁶¹Tb, ¹⁷⁷Lu, and ¹¹¹In. Suchagents may be complexed or otherwise bound, such bound directly to theadrenomedullin molecule or related derivative or chelated to theadrenomedullin related peptide through a chelator selected from:diethylenetriaminepentaacetic acid (DTPA),1,4,7,10-tetraazacyclododecan-N,N′,N″,N′″-tetraacetic acid (DOTA),1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′″-tetraacetic acid (TETA),and 6-hydrazinonicotinamide (HYNIC), among others.

EXAMPLE 1 A Process to Produce ^(99m)Tc-Labelled AM

In this example, the adrenomedullin produced is a human adrenomedullinhaving the sequence:

(SEQ ID NO.: 1) H-Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser- Pro-Gln-Gly-Tyr-15CONH₂

However, it is within the scope of the invention to use any othersuitable adrenomedullin, such as rat adrenomedullin or derivatives ofthe CT/CGRP/AMY peptide family, as well as their modified products suchas those obtained after N-terminal substitution, C-terminalsubstitution, or any other suitable substitution. In some embodiments ofthe invention, some of the amino acids are replaced by a non-amino acidmoieties, as described in further details hereinbelow.

A method for synthesizing an a CT/CGRP/AMY peptide suitable for use withthe present invention, such as for example adrenomedullin, was performedas follows. The following commercial N-α-fluorenylmethyloxycarbonyl[Fmoc]-L-amino acids were used: Alanine [Fmoc-Ala],Arginine-N^(ω)-(2,2,4,6,7-pentamethyidihydrobenzofuran-5-sulfonyl)[Fmoc-Arg(Pbf)], Asparagine-N^(γ)-trityl [Fmoc-Asn(Trt)], Asparticacid-α-t-butyl ester [Fmoc-Asp(OtBu)], Cysteine-S-trityl[Fmoc-Cys(Trt)], Glutamine-N^(δ)-trityl [Fmoc-Gln(Trt)], Glycine[Fmoc-Gly], Histidine-N^(im)-trityl [Fmoc-His(Trt)], Isoleucine[Fmoc-Ile], Leucine [Fmoc-Leu], Lysine-N^(ε)-t-butyloxycarbonyl[Fmoc-Lys(Boc)], Methionine [Fmoc-Met], Phenylalanine [Fmoc-Phe],Proline [Fmoc-Pro], Serine-O-t-butyl [Fmoc-Ser(tBu)],Threonine-O-t-butyl [Fmoc-Thr(tBu)], Tyrosine-O-t-butyl [Fmoc-Tyr(tBu)]and Valine [Fmoc-Val].

Adrenomedullin and its CT/CGRP/AMY analogues were synthesized, using asolid phase procedure based on the Fmoc-amino acid chemistry—BOP reagent(benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphoniumhexafluorophosphate) coupling strategy. This procedure is betterdescribed in reference 35, which is hereby incorporated by reference.

In summary, a Fmoc-Rink-amide-acetamidonorleucylaminomethyl resin(4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy-acetamidonorleucylaminomethylresin) was used as the solid support. After a treatment with a 20%piperidine (Pip)-dimethylformamide (DMF) mixture, in order to remove theprotecting Fmoc moiety and free the amine anchor on the solid support,the first amino acid of the synthesis, corresponding to the last residueof the peptide sequence (Tyrosine), was coupled to the resin with BOPreagent, in the presence of diisopropylethylamine (DIEA). In function ofthe resin substitution, a ratio of 3 equivalents (eq) of Fmoc-aminoacid, 3 eq of BOP and 5 eq of DIEA was used for each coupling step andeach step was monitored using a ninhydrin test.

After the complete synthesis of the peptide chain, a final Fmocdeprotection step was carried out with 20% Pip/DMF. For derivativescontaining a N-terminal chelating functional, the resin-bound peptidewas transferred into a round-bottom flask and the incorporation of aN-substituting moiety (examples of such moieties include, but are notlimited to, diethylenetriaminepentaacetic acid (DTPA) or1,4,7,10-tetraazacyclododecan-N,N′,N″,N′″-tetraacetic acid (DOTA) or1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′″-tetraacetic acid (TETA or6-hydrazinonicotinamide (HYNIC), among others) was achieved by mixingthe resin-adrenomedullin overnight, at 50° C., with 3 eq of thesubstituting compound, 3 eq of BOP reagent and 3 eq of DIEA dissolved ina solvent mixture of DMF-dichloromethane-dimethylsulfoxide (49%:49%:2%).

The peptide was finally cleaved from the resin using a mixture oftrifluoroacetic acid (TFA)-ethanedithiol (EDT)-phenol-H₂O (9.5 mL-0.25mL-0.3 g-0.25 mL; 10 mL/g of resin) for 2 hours, at room temperature.After TFA evaporation, the crude material was precipitated and washedusing diethylether. The peptide was then dried and kept at −20° C. untilcyclization and purification.

AM cyclization, purification and characterization was performed, asfollows. Crude adrenomedullin (AM) (200 mg) was dissolved in a 2 mLsolution of 50% dimethylsulfoxide/H₂O in order to generate the disulfidebridge between the cysteine side-chains. After 30 min at roomtemperature, the peptide solution was diluted with 500 mL of 10%acetonitrile (ACN) in aqueous TFA (0.06%) before being injected onto aRP-HPLC C₁₈ (15 μm; 300 Å) column (250×21.20 mm) (Jupiter column fromPhenomenex, Torrance, Calif.). The purification step was carried outusing a Waters Prep 590 pump system connected to a Waters Model 441absorbance detector. The flow rate was fixed at 20 mL/min and thepeptide was eluted with a solvent gradient of 0% to 100% solvent B, in 2h, where solvent A is 10% ACN in aqueous TFA (0.06%) and solvent B is45% ACN in aqueous TFA (0.06%).

The homogeneity of the various fractions was evaluated using analyticalRP-HPLC with a C₁₈ (5 μm; 300 Å) column (250×4.60 mm) ((Jupiter columnfrom Phenomenex, Torrance, Calif.) connected to a Beckman 128 solventmodule coupled to a Beckman 168 PDA detector. The flow rate was 1.0mL/min and the elution of the peptide was carried out with a lineargradient of 20 to 60% B, where A is TFA 0.06% and B is ACN. Aliquots of20 μl were injected and analyzed. Homogeneous fractions were pooled,lyophilized and then analyzed again by analytical HPLC, and by MALDI-TOFmass spectrometry (Voyager DE spectrometer—Applied Biosystems, FosterCity, Calif.) using α-cyano-4-hydroxycinnamic acid as a matrix forpeptide inclusion and ionization.

Labeling of a chelating adrenomedullin derivative (exemplified withDTPA-AM and ^(99m)technetium) was as follows. The substituted peptideDTPA-AM (18.5 μg-2.89 nmol) was dissolved in 1 mM HCl (100 μl) and then,SnCl₂.2H₂O (14.8 μl of a 0.2 mg/mL aqueous solution: 3 μg-13 nmol) wasadded, followed immediately with Na^(99m)TcO₄ (15 mCi-28.9 pmol) insaline. After 1 h at room temperature, the solution was diluted with 1mL of phosphate buffer-saline (PBS) at pH 7.4.

In other examples, adrenomedullin (AM) or AM fragments or AM analoguesare modified with agents able to bind radioactive and/or paramagneticchemical elements such as those from the following non limiting list:^(99m)technetium (^(99m)Tc), ¹¹¹indium (¹¹¹In), ⁶⁷gallium (⁶⁷Ga),⁶⁴copper (⁶⁴Cu), among others. In the present case, the modified AM, AMfragment or AM analogue is particularly suitable, but non-limitatively,for imaging with for instance, a gamma camera, a positron emissiontomography camera, a magnetic resonance instrument, or any othersuitable imaging device.

In other examples, adrenomedullin (AM) or AM fragments or AM analoguesare modified with agents able to bind radioactive elements such as thosefrom the following non exclusive list: ⁹⁰yttrium (⁹⁰Y), ¹⁶¹terbium(¹⁶¹Tb), ¹⁷⁷lutetium (¹⁷⁷Lu), ¹¹¹ indium (¹¹¹ In), among others. In thiscase, the modified AM, AM fragment or AM analogue are particularlysuitable, but non-limitatively, for application in radiotherapy.

In other examples, adrenomedullin (AM) or AM fragments or AM analoguesare modified with agents able to bind ions such as those produced fromthe elements appearing in the following non limiting list: iron (Fe),calcium (Ca), manganese (Mn), magnesium (Mg), copper (Cu), and zinc(Zn), among others. In the present case, the modified AM, AM fragment orAM analogues are particularly suitable, but non-limitatively, forapplication in chemotherapy, using, for example, an intracellular iondepletion strategy. In this particular case, possible chelating agentsfor ion depletion are selected form the following non-limiting list:desferioxamine, tachpyr (N,N′,N″-tris(2-pyridylmethyl)-cis-1,3,5-triaminocyclohexane), amongothers.

While a specific process for producing a labeled adrenomedullinderivative according to the invention is described hereinabove, thereader skilled in the art will readily appreciate that it is within thescope of the invention to produce labeled adrenomedullin derivativesthat are within the scope of the claimed invention in any other suitablemanner.

EXAMPLE 2 Pharmacokinetics of ¹²⁵I Labelled AM in Rats

Introduction

This example was designed to evaluate the biodistribution,pharmacokinetics and multiorgan clearance of AM in rats in vivo.Quantification of single-pass pulmonary kinetics of AM and its mechanismwas characterized further in dogs using the single bolusindicator-dilution technique.

Methods

All experimental procedures were performed in accordance with theregulations and ethical guidelines from Canadian Council for the Care ofLaboratory Animals, and received approval by the Animal Ethics andResearch Committee of the Montreal Heart Institute. Male Sprague-Dawleyrats (Charles River), weighing between 400-450 g, were anaesthetized byan initial intramuscular dose of xylazine (10 mg/kg of body weight) andketamine (50 mg/kg of body weight), followed by an intra-peritonealinjection of heparin (2000 units; Sigma). Catheters were inserted intothe right carotid artery and jugular vein. Heart rate and systemic bloodpressure were monitored continuously. Additional doses ofxylazine/ketamine were used if noxious stimuli (pinching the hind feet)elicited nociceptive motor reflexes or changes of the systemic bloodpressure. Venous and arterial blood samples (3 ml) were collected andcentrifuged (1875 g, 15 min, 4° C.) and the plasma saved for subsequentmeasurement of irAM (immunoreactive AM). A similar amount of saline wasinfused into the animals to prevent hypovolaemia.

Radiolabelled ¹²⁵I-rAM(1-50) (Amersham Biosciences) (¹²⁵I labelled ratadrenomedullin) was injected in a volume of 200 μl (0.3 pmol, 0.5 μCi)either into the right heart chambers via the right jugular vein catheter(n=10), or in the systemic circulation via the carotid catheter (n=10).A series of 200 μl blood samples were collected 1 min after the initialAM injection, then repeated every 5 min for a 30-min period. After eachcollection, an equal volume of saline was injected into the animal tomaintain blood volume and pressure. The animals were then killed and thelungs, liver, kidneys (en bloc with the adrenal glands) and heart wereremoved and gravity drained. The blood samples and organs were thenplaced in a gamma-counter (model 1470 Wizard; Wallac) to determine ¹²⁵Iradioactivity. Results are expressed as a percentage of totalradioactivity injected. Results for these experiments are shown in FIGS.1 and 2.

Effects of Antagonists on AM Clearance

Three groups of rats (n=20 in each) were studied and received either 200μl of hAM(22-52) (5.6 nmol; Bachem), 100 μl of CGRP (1.75 nmol; PhoenixPharmaceuticals) or 100 μl of unlabelled rAM(1-50) (17.5 nmol; AmericanPeptide). The drugs were given by either intraarterial (n=10 in eachgroup) or intravenous (n=10 in each group) injection 5 min before the¹²⁵I-rAM(1-50) bolus. Plasma samples and tissues were treated asdescribed above. Results are shown in FIG. 3.

Measurement of Endogenous rAM(1-50) Levels in Plasma and Tissues

Plasma levels were measured in samples obtained at baseline as describedabove (n=40). In order to evaluate endogenous tissue levels, tenadditional rats were studied. After being anaesthetized, the animalswere killed by removal of the lungs, liver, kidneys and heart.Homogenization of organs was performed by adding 2 ml of buffer [4 mol/lguanidine thiocyanate (Fisher Scientific) and 1% trifluoroacetic acid(Sigma)] to 200 mg of tissue samples with the use of an automaticrevolving pestle (DynaMix; Fisher Scientific). Homogenates werevortex-mixed and samples (100 μl) kept at 4 C for subsequent proteindetermination by Bradford analysis. Remaining samples were centrifugedat 1300 g (4 C) and the supernatant saved for processing. Tissues andplasma samples were extracted using Sep-Pak C18 cartridges (Waters) andirAM(1-50) was measured using a competitive RIA (PhoenixPharmaceuticals) according to the manufacturer's instructions. Thedetection limit of this assay is approx. 4.7 pg/tube with a specificityfor rAM(1-50) of 100%, without any cross reactivity (0%) with hAM(1-52),pro-hAM, pro-rAM, amylin and ET (endothelin)-1.

In Vivo Signal-Pass Measurement of am Clearance in Dogs

Dogs were anaesthetized and prepared as described in detail in reference[13], which is hereby incorporated by reference. A catheter was insertedinto the carotid artery and positioned just above the aortic valve. Thiscatheter was connected to a peristaltic pump for automated bloodwithdrawal. Another catheter was placed into the jugular vein andpositioned in the right ventricular outflow tract to allow bolusinjection of the study tracers. A bolus was prepared by adding 3.3 μCiof 125I-rAM(1-50) (2.9 pmol) to 3 ml of Evans-Blue-dye labelled albuminand 0.9% saline to give a final volume of 6 ml. The mixture wasseparated into three equal parts for the two successive experiments ineach animal and to realize dilution-curve standards. A baselinesingle-bolus indicator-dilution experiment was performed. After 5 min,CGRP(n=7), hAM(22-52) (n=8) and unlabelled rAM(1-50) (n=9) wereadministered as an intravenous bolus of 100 nmol and 5 min later asecond indicatordilution experiment was performed.

The collected samples were processed and indicator-dilution curvesconstructed and analyzed as described in previously cited reference 13.Cardiac output and mean tracer AM extraction during the pulmonarytransit time were computed from the curves. Mean tracer extractioncorresponded to the difference between the areas of the outflow curve ofthe vascular reference (albumin) and that of the extracted tracer (AM).Recirculation of the tracers apparent in the terminal portion of thecurves was removed by linear extrapolation of the semi-logarithmic downslopes. Results are shown in FIG. 4.

Statistical Analysis

Multiple group comparisons were performed by factorial ANOVA, followed,when a significant interaction was found, by the Bonferroni/Dunn t test.Plasma kinetics of 125I-rAM(1-50) was fitted using a two-phaseexponential decay equation with GraphPad Prism (version 4.0) software.Pulmonary clearance of 125I-AM(1-50) in rats after intravenous andintra-arterial injection was compared by two-tailed unpaired Student's ttest. Comparison between venous and arterial rAM(1-50) levels in plasmawas performed with a two-tailed paired Student's t test. In the canineexperiments, the effect of drugs on AM extraction was analyzed bytwo-tailed paired Student's t tests. A P value of <0.05 was consideredsignificant. All results are reported as means±S.E.M.

Results

Kinetics of ¹²⁵I-rAM(1-50) in Plasma

As shown in FIG. 1, intravenously administered ¹²⁵IrAM(1-50) rapidlydecreased following a two-compartment model with a relatively rapiddistribution half-life of 2.0 min [95% Cl (confidence interval),1.98-2.01] and an elimination half-life of 15.9 min (95% Cl, 15.0-16.9).Compartmental analysis revealed that the ratio of rate constants forexchange between the central and peripheral compartments (k₁₋₂/k₂₋₁) wasrelatively high at 7.97, demonstrating an important distribution of druginto the peripheral compartment. The volumes of distribution werecomputed, including the volume of the central compartment (Vc=3.84 ml),the volume at steady state (Vss=12.5 ml) and the apparent volume ofdistribution (Varea=35 ml). Administration of a human AM fragment(hAM(22-52)), CGRP or unlabelled rAM(1-50) prior to the injection ofradiolabelled rAM(1-50) did not modify plasma kinetics, resulting inalmost perfectly superimposable curves (results not shown).

Biodistribution of 125I-rAM(1-50) after Injection

As shown in FIG. 2, the lungs predominantly retained the peptide 30 minafter the injected dose (P<0.001). There was proportionately only minorretention by the liver, kidneys and heart. Administration of hAM(22-52)and CGRP did not significantly modify this distribution, except inkidneys, where only hAM(22-52) elevated the retained activity (P<0.005).Injection of unlabelled rAM(1-50) caused an important reduction in lungactivity (P<0.001) and significantly increased (P<0.001) the amountretained by the liver, kidneys and heart.

Lung retention after intravenous compared with intra-arterialadministration.

In the control group (n=10 per injection site), there was evidence ofimportant first-pass pulmonary retention with a more than 50% decline inthe amount of the peptide retained after intra-arterial compared withintravenous injection (FIG. 3). This pulmonary first-pass retention wasnot affected by prior administration of hAM(22-52) or CGRP. However,pre-treatment with rAM(1-50) did not decrease further the alreadylowered pulmonary retention after intra-arterial compared withintravenous injections.

Endogenous rAM(1-50) Levels in Plasma and Organs

There was no difference in I-rAM(1-50) levels in venous (3.1+−0.2pmol/l) and arterial (3.2+−0.2 pmol/l) plasma (n=40). Tissue levels(n=10) were more than 20-fold higher in the lungs (249.0+−48.3 pg/mgprotein; P<0.001) compared with liver (11.1+−1.3 pg/mg of protein),kidneys (11.7+−1.4 pg/mg of protein) and heart (7.2+−0.9 pg/mg ofprotein).

Single-Pass Pulmonary Kinetics of ¹²⁵I-rAM(1-50) in Dogs In Vivo

Analysis of the indicator-dilution curve outflow profiles demonstrated asignificant first-pass retention of ¹²⁵I-rAM(1-50). A typical experimentis shown in the FIG. 4(A). The curve for ¹²⁵I-rAM(1-50) progressivelydeviated from its vascular reference (labelled albumin). Therecirculation of tracers was removed by extrapolation of thesemi-logarithmic down slopes. The difference between the areas of thetwo tracers' curves, which represent mean tracer ¹²⁵I-rAM(1-50)extraction during a single pulmonary transit time, was 30% in thatexperiment. Plotting the natural log ratio of the two tracerscharacterized further the extraction over time (FIG. 4). Therelationship was found to be linear, demonstrating that extractionincreased over time with no evidence of return of the extracted peptideinto circulation. In terms of ordinary capillary modeling, the slope ofthis relationship represents the sequestration rate constant for¹²⁵I-rAM(1-50) by the lungs. In the same animal, a second experiment wasperformed after injection of unlabelled rAM(1-50) (FIG. 4B). There wasan evident reduction in pulmonary clearance with a smaller differentialcurve area compared with albumin (mean extraction 12%) and progressivelyconverging curves on the down slope. The log ratio is completelymodified with an initial plateau followed by a decrease, demonstratingthe return of the tracer into the circulation.

Mean single-pass pulmonary extraction of ¹²⁵IrAM(1-50) was 36.4±2.1%.This was significantly decreased (P<0.01) to 21.9±2.4% after theadministration of unlabelled rAM(1-50). Extraction was not affected byCGRP with 44.6±2.9% occurring in the control compared with 40.6±2.9%after administration. There was a slight but significant (P<0.01)decrease in extraction with hAM(22-52) from 40.0±1.7% before to31.4±3.3% after administration.

Discussion

In this study, plasma kinetics and biodistribution of exogenouslyadministered AM in rats as well as plasma and tissue levels ofendogenous AM were evaluated. Single-pass pulmonary clearance of AM indogs using the indicator-dilution technique was further quantified andcharacterized in vivo.

Injected AM has a relatively short elimination half-life of 16 min withrapid and important distribution into a peripheral compartment. Thelungs retain most of the injected activity with evidence of single-passclearance, since retention is lower after intra-arterial compared withintravenous injection. There was no difference in total endogenous I-rAMlevels across the pulmonary circulation with very high endogenous tissuelevels also found in the lungs compared with other organs. These datademonstrate that the lungs are a major site for AM clearance, theabsence of a gradient suggesting that the lungs also have the ability toproduce and release AM into the circulation.

A relatively small volume of distribution for the central compartment(3.5 ml) was found, which is, in fact, less than the total blood volumeof the rat. This is consistent with the very rapid clearance of AM fromplasma with evidence of a first-pass effect into the pulmonarycirculation. Thus a substantial proportion of intravenously injected AMis relatively rapidly cleared as it passes through the pulmonarycirculation and does not distribute into the systemic circulation. Theimportance of a first-pass pulmonary clearance was confirmed andquantified by the use of the indicator-dilution experiments in dogswhere it was found that approximately 36% of the injected AM wasretained within the few seconds of a single pulmonary transit time. Theoutflow profile demonstrates that the retained AM is bound to itsclearance site and does not return into the circulation. This, combinedwith the data in rats, would suggest that AM binds with relatively highaffinity and relatively irreversibly to its receptor. This is consistentwith previous data demonstrating important specific AM binding sites inthe lungs of rats and humans [15,16], with maximum binding in the lungsbeing higher than in any other organ studied [16]. This profile isreminiscent of the potent vasoconstrictor ET-1, which is alsopredominantly cleared by the pulmonary circulation by the endothelialETB receptor [13].

The effects of AM are mediated by at least two different receptors [17].One is the CGRP receptor to which AM binds with low affinity, whereasthe other is considered a specific AM receptor that can blocked by theC-terminal fragment of AM, hAM(22-52). In the present study, it wasfound that ¹²⁵I-rAM(1-50) clearance by the lungs can be competitivelyinhibited by the administration of unlabelled rAM(1-50). Interestingly,however, unlabelled rAM(1-50) did not modify the plasma kinetics of thepeptide, as we observed a compensatory increase in retention by theliver, kidney and heart. This supports further the important clearancerole of the lungs and suggests that most of the injected unlabelled AMwas also retained by the lungs, explaining the lack of inhibition inperipheral organs where levels of ¹²⁵I-rAM(1-50) must have been higherthan those of the unlabelled peptide. There was no effect of similardoses of CGRP, demonstrating that the CGRP receptor is not responsiblefor pulmonary clearance. Administration of the C-terminal fragmenthAM(22-52) also did not modify pulmonary retention in rats, although itdid cause a small significant increase in the kidneys. These resultswere confirmed by the in vivo indicator-dilution studies in dogs wherewe found important first-pass extraction of ¹²⁵I-rAM(1-50) which wasimportantly reduced after injection of unlabelled rAM(1-50), slightlyreduced after hAM(22-52) and unaffected by CGRP. Previous investigatorshave evaluated pulmonary clearance of AM in isolated rat lungs andpulmonary endothelial cells and found that AM levels in effluents andculture media were unchanged after CGRP, but increased afteradministration of hAM(22-52) [18]. The structural components of the CGRPand AM receptors, CRLR, RAMP1, RAMP2 and RAMP3 are all expressed in ratlungs [19]. Northern-blot analysis has revealed previously that RAMP2,which confers AM selectivity to the receptor, is highly expressed in ratlung tissues compared with RAMP1 and RAMP3 [20]. Using selective CRLRantibodies and immunohistochemistry, Hagner et al. [21,22] demonstratedintense staining in the alveolar capillaries of both humans and rats.These previous findings, together with the present study, suggest thatlung AM clearance is mediated by specific AM receptors, possibly at thelevel of the pulmonary vascular endothelium.

Conclusions

The lung is a primary site for AM clearance. There is importantfirst-pass pulmonary clearance of AM through specific receptors. Thissuggests that the lungs not only modulate circulating levels of thispeptide, but also represent its primary target.

EXAMPLE 3 Pharmacokinetics of ^(99m)Tc Labelled AM and Imaging UsingSame

Widely accessible in most nuclear medical centres via ⁹⁹Mo/^(99m)Tcgenerator. Technetium-99m shows suitable nuclear properties for nuclearimaging with γ-emitting of 140.5 keV and a short half-life of 6.01 h(34). To avoid strong perturbation of hAM1-52 chemical structure and,consequently, the loss of its biological properties duringradiolabelling with ^(99m)Tc, a successful procedure, called‘bifunctional approach’, has been proposed. This strategy consists oftethering a strong chelating group for the radionuclide to a point ofthe peptide that is irrelevant for preserving its biological properties[23, 26, 31]. Thus, we developed chelated radiolabelled adrenomedullinderivatives, preferably a chelated hAM1-52 derivative usingdiethylenetriaminepentaacetic acid (DTPA) radiolabelled with ^(99m)Tc.

The present example was designed to systematically evaluate thebiodistribution, pharmacokinetics and multi-organic clearance of^(99m)Tc-DTPA-hAM1-52 in dogs in vivo. Furthermore, the purpose of thisinvestigation was to assess the utility of the radiolabelled peptide asa pulmonary vascular imaging agent.

Anesthesia and Animal Preparation

All experimental procedures were performed in accordance withregulations and ethical guidelines from Canadian Council for the Care ofLaboratory Animals, and received approval by the animal ethics andresearch committee of the Montreal Heart Institute. Mongrel dogsweighing between 20-30 kg and presenting negative Dirofilaria imitisblood test results were anesthetized by an initial intravenous dose ofpentobarbital sodium (50 mg/kg). Animals were intubated and mechanicallyventilated using room air. Cutaneous electrocardiographic leads wereinstalled, and 18 F cathlon with three-way was installed on bothsaphenous vein for 0.9% sodium chloride perfusion, radiolabelledinjection and blood collection. A right arterial femoral catheter wasalso inserted using the Seldinger technique for continuous bloodpressure monitoring. Additional doses of pentobarbital sodium were usedif noxious stimuli (pinching near the eye) could elicit nociceptivemotor reflexes or changes of the systemic blood pressure.

Dogs (n=10) undergoing surgical procedures were anesthetized andprepared as previously described, but maintained ventilated with 1-3%isoflurane. Pulmonary lobectomy was obtained by performing a surgicalligature of the right median lobe of the lungs.

Pharmacokinetics of 99mTc-DTPA-hAM1-52 in Plasma

Purified and buffered ^(99m)Tc-DTPA-hAM1-52 samples were injected inright saphenous vein (n=6). A series of 2 mL blood samples werecollected 1 min after the initial AM injection for a 10-min period, thenrepeated every 5 min for the following 50-min period. Blood samples weretaken via left saphenous vein. After each collection, an equal volume ofsaline was injected into the animal to maintain blood volume andpressure. The blood samples were then placed in an automatic gammacounter (model 1272 Clinigamma, LKB Wallac, Finland) to determine^(99m)Tc activity. Results were expressed as a percentage of totalradioactivity injected per mL and are shown in FIG. 5.

Biodistribution of 99mTc-DTPA-hAM1-52 and Multi-Organic Clearance InVivo

Multi-organic biodistribution of ^(99m)Tc-DTPA-hAM1-52 was evaluatedwith an Anger camera (420/550 Mobile Radioisotope Gamma Camera;Technicare, Solon, Ohio, USA) equipped with on board computer, and alow-energy parallel-hole collimator (model 14S22014). Followingintravenous injection of ^(99m)Tc-DTPA-hAM1-52, dynamic acquisition ofthe lungs, heart, liver and kidneys was recorded for a 30-min period(one frame/sec during the first minute, then one frame/min for theremaining time). Static acquisitions was also recorded for wholeindividual organs, including lungs, kidneys, liver, heart, bladder,gallbladder and muzzle, at 30, 60, 120, 240 minutes after initialinjection. These recordings were performed both in ventral and dorsalpositions. Results are shown in FIGS. 6 and 7.

Gamma Camera Results Analysis

Dynamic and static acquisitions were evaluated by using Matlab version7.01 image analysis tools software. The ^(99m)Tc total count, ^(99m)Tcmean count, and region of interest (ROI) size were calculated for eachorgan. Data correction was applied for 1) radioactive decay, 2) surgicaltable attenuation (dorsal images only), 3) geometric mean, and 4)organ's attenuation based on transmission factor. Results were expressedas a percentage of total radioactivity injected and examples of imagesobtained are shown in FIGS. 8 and 9.

Statistical Analysis

Plasma kinetics of ^(99m)Tc-DTPA-hAM1-52 was fitted using a two-phaseexponential decay equation with GraphPad Prism version 4.0 software.Time effects on each organ biodistribution were analyzed by two-wayrepeated measures ANOVA followed, when a significant interaction wasfound, by Bonferroni/Dunn t-test. Multiple organs biodistributioncomparison at 30 minutes was performed by one-way ANOVA followed, when asignificant interaction was found, by Bonferroni/Dunn t-test. A P valuesof <0.05 was considered significant. All results are reported asmean±S.D.

Results

Kinetics of ^(99m)Tc-DTPA-hAM1-52 in Plasma (FIG. 5)

Intravenously administered ^(99m)Tc-DTPA-hAM1-52 decreased relativelyrapidly following a two-compartment model with a relatively rapiddistribution half-life of 1.75 min (95% confidence interval, Cl:1.31-2.65) and an elimination half-life of 42.14 min (Cl: 30.41-68.63).Compartmental analysis reveals that the ratio of rate constant forexchange between the central and peripheral compartments (k₁₋₂/k₂₋₁) isrelatively high at 24.09, demonstrating an important distribution ofdrug into the peripheral compartment.

Biodistribution of ^(99m)Tc-DTPA-hAM1-52 after Injection (FIG. 6)

The lungs predominantly retained the peptide with 27.00±2.76% of theinjected dose after 30 minutes (P<0.001), as compared to kidneys(19.17±3.06%), liver (11.67±1.37%), heart (7.17±2.04%), bladder(5.67±1.75%), gallbladder (0.96±0.38%), and muzzle (1.17±0.41%). Lungretention was mildly reduced with time but sustained up to 4 hours afterthe injection (15.83±2.32%). Furthermore, uptake progressively increasedin the bladder (26.83±4.36%) and gallbladder (0.83±0.41%), consequentlyto the excretion of the radiolabelled peptide. The ^(99m)Tc-DTPA-hAM1-52biodistribution in the kidneys, liver, and muzzle remained unchangedwith time, with respectively 20.67±1.51%, 10.67±1.75%, and 0.83±0.41% at240 minutes after peptide injection.

Dynamic Bioditribution ^(99m)Tc-DTPA-hAM1-52 after Injection (FIG. 7)

Analysis of dynamic multi-organic biodistribution demonstratessignificant pulmonary first pass retention of ^(99m)Tc-DTPA-hAM1-52. Thecurve for lungs clearance also shows recirculation of the radiolabelledpeptide, followed by a slow decrease with time. Moreover, heart curveindicates similar first pass retention of ^(99m)Tc-DTPA-hAM1-52, withouthowever sustained clearance with time. On the opposite, liver andkidneys dynamic biodistribution demonstrate only slow but continuousretention with time.

Selective Pulmonary Lobectomy Effects on ^(99m)Tc-DTPA-hAM1-52 Perfusion(FIGS. 8 and 9)

Homogeneous distribution of the tracer is evident in the lungs of anormal animal (FIG. 8) with substantially no detectable activity overthe region of the heart and little activity in the abdomen. This allowsfor good lung imaging without significant contaminant activity fromsurrounding organs. After surgical obectomy mimicking the pathologiccondition of a pulmonary embolus (FIG. 9), there is an evident perfusiondefect which allows the diagnosis by external imaging. FIG. 9 showsimages obtained through anterior (panel A) and oblique (panel B) views.The perfusion defect was substantially wedge-shaped. This defect isindicated by an arrow and substantially delimitated by dotted lines.

EXAMPLE 4 Synthesis of Alternative Adrenomedullin Derivatives

Adrenomedullin and adrenomedullin fragments were synthesized as follows.These peptides were synthesized according to a procedure based onstandard solid phase Fmoc peptide chemistry. Briefly, a Rink amide AMresin was used as the solid support and all couplings of. N-α-Fmoc aminoacids were performed in N,N-dimethylformamide (DMF) in the presence ofbenzotriazol-1-yl-oxy-tris(dimethylamino)-phosphoniumhexafluorophosphate (BOP) and diisopropylethylamine (DIEA). Completionof the reaction was monitored using a ninhydrin test. Once the peptidechain was completed, cleavage from the resin was achieved with a 2 htreatment with a 95% trifluoroacetic acid (TFA) solution containingethanedithiol, phenol, and water as scavengers. The solid support wasthen removed by filtration and, after TFA evaporation, the crude peptidewas isolated by precipitation with diethylether. All chemicals usedduring the syntheses were from known suppliers.

The crude material was dissolved in water containing 0.06% TFA, at aconcentration of 10 mg/ml. Dithiothreitol (5 eq) was added to ensurecomplete linearization of the peptide. This solution was purified bymeans of reverse-phase HPLC using a C18 (5 μm, 110 Å, 250×21.2 mm)column and the peptide detection was carried out with a UV detector setat 229 nm. Elution was achieved over a 2 h linear gradient from A (watercontaining 0.06% TFA) to B (40% acetonitrile in A). The flow rate wasmaintained at 20 ml/min. Collected fractions were evaluated for theirpurity by analytical reverse-phase HPLC with a C18 (4 μm, 90 Å, 250×4.6mm) column connected to a photodiode array detector. The flow rate wasmaintained at 1 ml/min and the elution was carried out with a 1 h lineargradient of 0% to 60% acetonitrile in aqueous 0.06% TFA. Homogeneousfractions were analyzed by MALDI-TOF mass spectrometry. Analyses wereperformed with a nitrogen laser (337 nm) and α-cyano-4-hydroxycinnamicacid was the matrix for ionization. Each mass spectrum was recorded inlinear mode at an accelerating voltage of 25 kV. Fractions correspondingto pure linear AM were pooled, lyophilized and kept at −20° C. untilfurther use.

A small amount of pure linear AM was dissolved in water at aconcentration of 1 mg/ml and aliquots of 17.4 μl (2.89 nmol) were placedat the bottom of 2 ml sterile tubes. Aliquots were then frozen,lyophilized and kept at −20° C. until the ^(99m)Tc labelling procedure,which was as follows.

Labeling of linear hAM(1-52) was realized using ^(99m)Tc by a directmethod. 100 ml of 1 mM hydrochloric acid was added to a reaction vialcontaining 18.5 mg of lyophilized linear hAM(1-52). Immediatelythereafter, 14.8 mL of freshly prepared SnCl2 (0.2 mg/mL) solution wasadded. After addition of 0.2 ml of freshly eluted 99mTc-sodiumpertechnetate (80-100 mCi/ml saline), the mixture was gently stirred andincubated for 1 h at room temperature.

In this and all the following examples, the adrenomedullin used is ahuman adrenomedullin, a fragment thereof, or a derivative of the humanadrenomedullin having the sequence:

(SEQ ID NO.: 1) H-Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser- Pro-Gln-Gly-Tyr-CONH₂.

However, in alternative embodiments of the invention, the adrenomedullinis any other suitable mammalian adrenomedullin, such as rat, mouse, dogor other suitable mammalian adrenomedullin.

As compared to the experiments presented in examples 1 to 3, it has beenfound that linear adrenomedullin is able to directly bind to ^(99m)Tc.It is hypothesized that ^(99m)Tc binds to the molecule throughcoordination bonds involving the sulphur atoms of the free thiolfunctions of cysteines 16 and 21 of the above-described hAM. This typeof bonding was described previously for salmon calcitonin, afterreducing the disufide bridge (Bioconjugate Chem, 16, 939-948 [2005]). Inaddition, it is assumed that the nitrogen atoms of the peptide bond ofboth cysteines also participate the chelation process.

EXAMPLE 5 Bio-Distribution of ^(99m)Tc-Linear AM

The bio-distribution of ^(99m)Tc-linear AM, synthesized as described inExample 4, was studied in dogs as a function of time in various organs.^(99m)Tc-linear AM was injected in dogs and multi-organicbiodistribution of ^(99m)Tc-DTPA-(synthesized as described hereinabove)was evaluated with an Anger camera (420/550 Mobile Radioisotope GammaCamera; Technicare, Solon, Ohio, USA) equipped with on board computer,and a low-energy parallel-hole collimator (model 14S22014). Followingintravenous injection of linear ^(99m)Tc hAM1-52, dynamic acquisition ofthe lungs, heart, liver and kidneys was recorded for a 30-min period(one frame/sec during the first minute, then one frame/min for theremaining time). Static acquisitions were also recorded for wholeindividual organs, including lungs, kidneys, liver, heart, bladder,gallbladder and muzzle, at 30, 60, 120, 240 minutes after initialinjection. These recordings were performed both in ventral and dorsalpositions. Dynamic and static acquisitions were evaluated by usingMatlab version 7.01 image analysis tools software. The ^(99m)Tc totalcount, ^(99m)Tc mean count, and region of interest (ROI) size werecalculated for each organ. Data correction was applied for 1)radioactive decay, 2) table correction (dorsal images only), 3)geometric mean, and 4) organ's attenuation based on transmission factor.Results were expressed as a percentage of total radioactivity injected.

As seen from FIG. 10, ^(99m)Tc-linear AM binds relatively selectively tothe lungs with 24% retention and is only relatively slowly eliminatedfrom this organ. These results are similar to results obtained usingcyclic adrenomedullin presented in the previously referred to PCTapplication. Knowing that reduced linear AM is a weak ligand(Endocrinology, 135, 2454-2458 [1994]), these results suggest that theincorporation of ^(99m)Tc between the sulfur atoms of the cysteineside-chains promotes the folding of the 16 to 21 segment of the moleculein a similar pattern to that found in native AM. Therefore, it can bepostulated that labeled linear adrenomedullin and derivative thereofhave a potential to be useful in the imaging of the lungs.

It should be noted that ¹²⁵I, which is often use in pre-clinicalbiodistribution studies, is a radioactive element of practically noclinical value in human imaging due to its weak radioactive activity andpotential thyroid toxicity. Therefore, the surprising result that someradioactive elements, such as ^(99M)Tc, readily bind to linear AMindicates that linear AM is likely to be successfully used in lungimagery.

EXAMPLE 6 Plasma Kinetics of ^(99m)Tc-Linear AM

The plasma kinetics of ^(99m)Tc-linear AM was studied in 11 rats.^(99m)Tc-linear AM was injected in rats in a volume of 200 μl into theright jugular vein catheter. A series of 200 μl blood samples werecollected 1 min after the initial injection, then repeated every 5 minfor a 30-min period. After each collection, an equal volume of salinewas injected into the animal to maintain blood volume and pressure. Theanimals were then sacrificed and the lungs, liver, kidneys (en bloc withthe adrenal glands) and heart were removed and gravity drained. Theblood samples and organs were then placed in a gamma counter (model 1470Wizard, Wallac, Finland) to determine ^(99m)Tc activity. Results wereexpressed as a percentage of total radioactivity injected.

As seen from FIG. 11, ^(99m)Tc-linear AM is cleared relatively rapidlyfrom the plasma. The data illustrated in FIG. 11 was used to determinethe parameters of two-compartment model, which gave a relatively rapiddistribution half-life of 0.54 min and an elimination half-life of 5.88min.

As seen in FIGS. 12 and 13, which illustrate the biodistribution of^(99m)Tc-AM in rats for various organs, ^(99m)Tc-linear AM issignificantly retained by lungs, the lung uptake being maintained for atleast 1 hour, thereby enabling external imaging. ^(99m)Tc-linear AM iseliminated by the kidneys (mostly) and the liver. Furthermore, thyroiduptake was minimal, which indicates that this compound is likely to berelatively safe for human use.

As seen in FIG. 14, labeling efficiency of purified ^(99m)Tc linearhAM1-52 enabling potential human use can be obtained by mini-columnpurification. To evaluate amount of purified ^(99m)Tc linear hAM1-52,colloids and unlabelled ^(99m)Tc, instant thin layer chromatography onsilica gel impregnated glass fiber paper (ITLC SG) (P/N 61886, Pall LifeSciences) was performed on 1) radiolabelled solution before mini-columnpurification, and 2) sample obtained after C18 mini-column purification.ITLC SG-solvents were acetone (Fisher Scientific) for dosage ofunlabelled 99mTc, and BAPE solution (30 U butanol; 6 U acetic acid; 24 Upyridine; 20 U nanopure water) for colloids evaluation. The ^(99m)Tclinear hAM1-52 migrated only with the BAPE mixture. ^(99m)Tc activitywas assessed using an automatic gamma counter (model 1272 Clinigamma,LKB Wallac, Finland). Average radiochemical purity (% of99mTc-DTPA-hAM1-52) was 65% prior to column separation, compared to 91%after a first column purification and 94% after a second columnpurification. For all experiments greater than 90% purification wasused.

FIG. 15 illustrates the stability of purified ^(99m)Tc linear hAM1-52 atroom temperature as verified by ITLC after 24 and 48 hours. The productis stable and retains greater than 90% purity after 48 hours inphosphate buffered saline (PBS). Such stability is important anddesirable as product for clinical imaging may require delay betweenpreparation and injection.

FIG. 16 illustrates in vivo single-pass measurement of purified ^(99m)Tclinear hAM1-52 clearance in dogs. Dogs were anesthetized and a catheterwas inserted into the carotid artery and positioned just above theaortic valve. This catheter is connected to a peristaltic pump forautomated blood withdrawal. Another catheter is placed into the jugularvein and positioned in the right ventricular outflow tract to allowbolus injection of the study tracers. A bolus was prepared by adding 2mCi of purified ^(99m)Tc linear hAM1-52 to 3 ml of Evans blue dyelabeled albumin and 0.9% saline for a final volume of 6 ml. A singlebolus indicator-dilution experiment was performed. The collected sampleswere processed and indicator-dilution curves constructed and analysed.Mean tracer ^(99m)Tc linear hAM1-52 AM extraction during the pulmonarytransit time was computed from the curves. Mean tracer extractioncorresponds to the difference between the areas of the outflow curve ofthe vascular reference (albumin) and that of the extracted tracer(^(99m)Tc linear hAM1-52). Recirculation of the tracers apparent in theterminal portion of the curves is removed by linear extrapolation of thesemi-logarithmic down slopes.

FIG. 16 demonstrates significant single pass extraction of purified^(99m)Tc linear hAM1-52 by the dog lung with a mean extraction of 21% inthis example.

EXAMPLE 7 Affinity of Various Human Adrenomedullin (hAM) Derivativeswith the Lungs

FIGS. 17A, 17B and 17C illustrate the capability of various humanadrenomedullin (hAM) derivatives (amino acids contained in each fragmentmentioned in parenthesis) in displacing iodine-marked humanadrenomedullin in the lungs of dogs. These Figures illustrate that thereis clearly a relatively well-defined concentration of the differentpeptides that allow to displace 50% of the human adrenomedullin in thelungs. These concentrations, denoted by IC-50, are shown in Table 1 forvarious adrenomedullin fragments and linear and cyclic adrenomedullinpeptides.

TABLE 1 IC₅₀of various adrenomedullin derivatives. Synthetic PeptidesIC₅₀ hAM (cyclic) 5.5 × 10⁻¹¹M hAM linear 7.1 × 10⁻⁹M N-Ac hAM 4.1 ×10⁻¹¹M hAM(1-25) ≧10⁻⁵M hAM(13-52) 3.4 × 10⁻⁸M hAM(22-52) 8.1 × 10⁻⁷MhAM(26-52) ≧10⁻⁶M hAM(40-52) ≧10⁻⁵M hAM2 4.8 × 10⁻⁷M hAM2(16-47) 9.8 ×10⁻⁷M hCGRP 4.4 × 10⁻⁷M CGRP(8-37) ≧10⁻⁵M PAMP ≧10⁻⁵M

Table 1 clearly shows that linear adrenomedullin (hAM linear) bindsrelatively well to the receptors to which cyclic adrenomedullin binds inthe lungs of dogs. Indeed, the IC-50 of linear hAM is only two orders ofmagnitude larger than the IC-50 of cyclic hAM. Therefore, linearadrenomedullin, which is relatively easily synthesized and allows tobind some radioactive elements thereto, such as ^(99M)Tc, may be used inlung imagery for various applications.

N-terminal acetylated AM (N-Ac hAM) did not seem to affect the bindingof hAM to the lungs of dogs. Also, the results from these bindingexperiments of peptides from the CT family correspond to the bindingprofile for the AM1 (CRLR+RAMP2) receptor: AM>AM (13-52)>CGRP and AM(22-52)>CGRP (8-37).

AM2, a peptide within the same family as AM has an affinity similar toCGRP for the lung of dogs. It is likely that AM2 binds to a complexincluding CRLR and another RAMP protein.

In addition, binding of L-AM and C-AM was evaluated by using a humanbreast adenocarcinoma cell line (MCF-7). These cells expressapproximately 50,000 AM receptors per cell. For each peptide,competition binding experiments were performed in triplicates using¹²⁵I-AM(1-52). Both C-AM and L-AM displayed competitive binding on MCF-7cells with IC50 of 19.6 nM and 70.3 nM respectively.

EXAMPLE 8 Imaging of the Lungs with Purified ^(99m)Tc Linear hAM1-52

FIGS. 18 and 19 illustrate respectively images of the lungs of a dog anda rat to which purified ^(99m)Tc linear hAM1-52 has been administered.In both species there is relatively specific and homogeneous pulmonaryuptake, thereby enabling good external imaging.

EXAMPLE 9 Bio-Distribution of Cyclic and Linear ^(99m)Tc-AM in Dogs

The bio-distribution of cyclic and linear ^(99m)Tc-AM was studied indogs as a function of time in various organs. Cyclic or linear^(99m)Tc-AM was injected in dogs and multi-organic biodistribution of99mTc-DTPA-hAM1-52 was evaluated with a gamma camera. Followingintravenous injection, dynamic acquisition of the lungs, heart, liverand kidneys was recorded for a 30-min period (one frame/sec during thefirst minute, then one frame/min for the remaining time). Staticacquisitions was also recorded for whole individual organs, includinglungs, kidneys, liver, heart, bladder, gallbladder and muzzle, at 30,60, 120, 240 minutes after initial injection. These recordings wereperformed both in ventral and dorsal positions. Dynamic and staticacquisitions were evaluated by using Matlab version 7.01 image analysistools software. The ^(99m)Tc total count, ^(99m)Tc mean count, andregion of interest (ROI) size were calculated for each organ. Datacorrection was applied for 1) radioactive decay, 2) table correction(dorsal images only), 3) geometric mean, and 4) organ's attenuationbased on transmission factor. Results were expressed as a percentage oftotal radioactivity injected.

As seen from FIG. 20, cyclic and linear ^(99m)Tc AM binds relativelyselectively to the kidneys with between 20 and 30% retention and areonly slowly eliminated from this organ as this amount of retention ismaintained at 120 min. These results are surprising as linearadrenomedullin is the equivalent of a denatured protein and it could beexpected that such a derivative of adrenomedullin could only weakly bindto adrenomedullin specific receptors in any of the organs studied inthis experiment. Therefore, these results suggest that not only cyclicbut also labeled linear adrenomedullin and derivative thereof have apotential to be useful in the imaging of the lungs.

EXAMPLE 10 Plasma Kinetics of ^(99m)Tc-Cyclic AM and ^(99m)Tc-Linear AM

The plasma kinetics of ^(99m)Tc-cyclic AM and ^(99m)Tc-linear AM werestudied in 7 and 6 dogs respectively. The radiolabelled AM derivativeswere injected in a volume of 1.5 mL into a right jugular vein catheter.A series of 200 μl blood samples were collected 1 min after the initialinjection, then repeated every 5 min for a 30-min period. After eachcollection, an equal volume of saline was injected into the animal tomaintain blood volume and pressure. The blood samples were then placedin a gamma counter (model 1470 Wizard, Wallac, Finland) to determine^(99m)Tc activity. Results were expressed as a percentage of totalradioactivity injected.

As seen from FIG. 21, ^(99m)Tc-cyclic and ^(99m)Tc-linear AM are clearedrelatively rapidly from the plasma. The data illustrated in FIG. 21 wasused to determine the parameters of two-compartment model, which gave arelatively rapid distribution half-life of less than two min and anelimination half-life of less than 45 minutes for both derivatives.

As seen in FIG. 22 which illustrates whole body imaging of^(99m)Tc-linear AM in a dog 120 min after injection, ^(99m)Tc-linear AMis significantly retained by the kidney enabling good quality externalimaging. The images were acquired using a Siemens dual hear Ecam Gammacamera 120 min after injection of the compound. Anterior and posteriorviews are seen on the left and center while an attenuation image(similar to an x-ray) is demonstrated on the left. It is also notablethat urinary bladder activity is also easily seen as the tracer isexcreted by the kidneys.

FIG. 23 illustrates tomographic slices of the kidney from the sameanimal in transverse (first row), sagittal (middle row) and frontalsections (bottom row). ^(99m)Tc-linear AM is seen to concentrate in thekidney cortex, the kidney medulla being free of activity. This confirmsthat the distribution of AM binding sites are located at the kidneycortex and that the tracer could specifically be used to imageconditions known to affect cortical kidney function.

EXAMPLE 11 Kidney Imaging

Kidney damage was induced by a single intraperitoenal injection ofmonocrotaline 60 mg/kg in rats. Three weeks later the animal receivedwere anaesthetized and mechanically ventilated. An intravenous injectionof 800 μci ^(99m)Tc-linear AM was performed by the jugular vein andreceived and intravenous and 30 minutes later the activity retained bythe kidneys was determined by two different approaches: first byexternal imaging using a dual-hear Siemens Ecam gamma camera by drawingregions of interest around the kidneys, and second, after the animalswere sacrificed and their kidneys removed and counted in a gamma counterto determine ^(99m)Tc activity. By both methods, the activity in thekidneys was expressed as a percentage of the total activity injected.

FIG. 24 is a bar chart representing kidney activity in control rats(n=4) and in monocrotaline treated rats (n=5) after 3 weeks after kidneyinjury using external detection by an Ecam. Kidney uptake of the tracerwas reduced by more than half form 23%±3% to 10%±6% after monocrotalineinjury. This was concordant with the external counting of the organs(27%±6% and 11%±7% respectively). The use of labelled AM derivativestherefore allows for the detection of kidney damage in this animalmodel, and in other subjects, for example by integrating the detectedradioactivity over a kidney obtained by imaging the kidney in a subjectin which radioactively labelled AM or AM derivative has been injectedand comparing this integrated radioactivity with a baselineradioactivity obtainable in healthy subjects.

EXAMPLE 12 Imaging of Pulmonary Arterial Hypertension

AM-L Synthesis and Purification

Linear adrenomedullin (AM-L) was synthesized using a solid phaseprocedure based on a fluorenylmethyloxycarbonyl (Fmoc) chemistry with aRink-AM-amide resin (Chem-Impex International, IL, USA) as the solidsupport. N-α-Fmoc protected amino acids (Matrix Innovation Inc., QC,Canada) were introduced in the peptide chain following abenzotriazol-1-yl-oxy-tris(dimethylamino)-phosphoniumhexafluorophosphate (BOP) coupling strategy and each coupling reactionwas monitored to confirm its completion. Cleavage from the solid supportto obtain crude peptide was achieved with a mixture of trifluoroaceticacid (TFA)/ethanedithiol/water (92.5:2.5:2.5; 20 ml/g). After TFAevaporation, the peptide was precipitated using diethylether.

AM-L was purified by reversed phase-HPLC (RP-HPLC) using a FlangedMODCOL column (25×3.5 cm) packed with a Jupiter C18 matrix (15 μm, 300Å) (Phenomenex, Calif., USA). The purification step was carried outusing a Varian ProStar system at a flow rate maintained at 20 ml/min.The UV-Vis detector was set at 220 nm and the peptide was eluted fromthe column with a 2 h gradient from 10% to 50% ACN/H2O containing 0.06%TFA.

The purity of the collected fractions was evaluated through analyticalRP-HPLC and the mass was established with MALDI-TOF mass spectrometry(Voyager DE, Applied Biosystems, CA, USA). Homogeneous fractionscorresponding to the desired peptide were then pooled and aliquoted in2.9 nmol samples, before lyophilization.

Radiolabelling and Purification

Sample vials containing 2.9 nmol AM-L were kept at −20° C.Radiolabelling was performed by adding to the vial 100 μL of HCl 1 mM,and 14.8 μL of SnCl2.2.H₂O (0.2 mg/mL-13 nmol). Immediately afterdissolving the material, 15 mCi of freshly prepared Na^(99m)TcO₄ (28.9pmol) in saline solution was added and the mixture was kept at roomtemperature for 1 h. Following the radiolabelling step, 1 mL of PBS (pH7.4) was added to the solution.

The totality of the ^(99m)Tc-AM-L reaction mixture was injected onto a 1cc (100 mg) C18 Sep-Pak cartridge. The cartridge was then washed with 3mL of 1 mM hydrochloric acid and eluted with 3 mL of a 50% ethanolsolution. Fractions of 0.5 mL were collected into sterile polypropylenetubes. Fractions and cartridge radioactivity count was then measured andthree fractions with the highest counts were pooled. 200 μL of 10×sterile PBS (pH 7.4) was added and the radiochemical purity measured byinstant thin layer chromatography using ITLC-SG strips from PALL LifeSciences (PALL Corp.) was ≧95%.

Studies in Monocrotaline (MCT)-Induced Pulmonary Arterial Hypertension

Male Sprague Dawley rats weighting between 200-225 g received an 0.5 mLintraperitoneal (IP) injection of either 0.9% saline or 60 mg/kgmonocrotaline (MCT). Five weeks later, rats were anesthetized forhemodynamic measurements using Millar microtip pressure transducercatheters.

Nuclear Medicine Experiments.

The animals were anesthetized by an initial intra-muscular dose ofxylazine (10 mg/kg) and ketamine (50 mg/kg), followed by anintraperitoneal injection of heparin (2000 U, Sigma Chemical Co.).Additional doses of xylazine/ketamine were used if noxious stimuli (hindfeet pinching) could elicit nociceptive motor reflexes or changes of thesystemic blood pressure.

99mTc-AM-L was injected in a volume of 200 μl (0.3 pmol, 0.5 mCi) intothe right jugular vein. A series of 200 μl blood samples were collected1 and 3 min after the initial AM injection, then repeated every 5 minfor a 30-min period. After each sample collection, an equal volume ofsaline was injected into the animal to maintain blood volume andpressure.

The whole body biodistribution of radiolabelled peptide was evaluatedusing two different approaches: in vivo by imaging with a gamma camerasystem, and ex vivo by surgically removing and counting organs in agamma counter. In vivo multi-organic biodistribution of ^(99m)Tc-AM-Lwas evaluated with a Siemens E. Cam signature camera system equippedwith on board computer, and a low-energy parallel-hole collimator.Following intravenous injection of ^(99m)Tc-AM-L, dynamic acquisitionwas recorded for a 30-min period (one frame/sec during the first minute,then one frame/min for the remaining time). Static acquisitions werealso recorded for whole individual organs, including lungs, kidneys,liver, heart and bladder at 30 minutes after initial injection. At theend of in vivo acquisition, the animals were sacrificed and the lungs,liver, kidneys and heart (separated into right ventricle, leftventricle+septum) were removed, gravity drained and weighted. The bloodsamples and organs were then placed in a gamma counter (model 1470Wizard, Wallac, Finland) to determine ^(99m)Tc activity. Results wereexpressed as a percentage of total radioactivity injected.

Molecular Biology Experiments.

To perform lung protein extraction, the snap frozen right inferior lobewas homogenized using a polytron homogenizer in lysis buffer containinga protease inhibitor cocktail. The homogenate was clarified bycentrifugation and the final protein concentration was determined. Fiftymicrograms of protein per sample were separated on a 15% SDS-PAGE gelfor 1 hour at 200V at 40 C and transferred onto a polyvinylidenedifluoride membrane at 100V for 90 min at 40 C. The membrane wassubsequently blocked for 2 hours at room temperature with 5% skimmedmilk powder in PBS 1× and 0.01% tween 20 (PBS-T) and incubated overnightat 40 C with primary rabbit polyclonal antibody raised against aminoacids 28-166 of RAMP2 of human origin (Santa Cruz). The antibody wasdiluted 1:500 with 5% milk in PBS-T overnight at 40 C. The membrane wasthen washed with PBS-T and re-blocked for 10 min with 5% milk diluted inPBS-T. The membrane was then incubated with the appropriate horseradishperoxiduse-conjugated secondary antibody for rabbit (JacksonLaboratories) diluted 1:10000 in 5% milk PBS-T. Following three washes,the immunoreactive bands were visualized by enhanced chemiluminescence(Renaissance Plus, Perkin Elmer Life Sciences) according to themanufacturer's instruction using Bio-Max MR film. Anti-Actin 1:1000antibody was used as the housekeeping gene.

Statistical Analysis

Differences between groups were evaluated by two-tailed independentsamples t-tests. Plasma kinetics of ^(99m)Tc-AM-L were analyzed using atwo compartments pharmacokinetic model with Prism v4.0 software and thefitted curves were compared using an F test. All values are reported asmeans±standard deviations.

Results

MCT rats developed severe PAH with right ventricular systolic pressureof 88±26 mmHg (n=11) compared to 30±7 mmHg (n=8) in controls, P<0.001.There was also important right ventricular hypertrophy evidenced byhigher right to left ventricular+septum weight ratio of 0.50±0.07compared to 0.22±0.07, P<0.001.

Plasma kinetics of ^(99m)Tc-AM-L are presented in FIG. 25. The fittedcurves were significantly different (P<0.001) with plasma levelsapproximately two-fold higher in PAH compared to control animals. After10 minutes, levels were 1.25±0.28% the injected dose (ID) in controlscompared to 2.08±0.65% ID in PAH animals (P=0.03).

The biodistribution of ^(99m)Tc-AM-L 30 minutes after injection wasdetermined by two different approaches: in vivo by using a gamma camera(FIG. 26) and ex vivo by counting the explanted organs in a gammacounter (FIG. 27). The in vivo biodistribution revealed a markedlyreduced lung uptake of the tracer from 14±1% ID in controls to 4±1% inPAH, P<0.0001. A similar retention and reduction was observed ex vivowith 11±2% ID vs. 3±1%, P<0.001. The MCT treated group also demonstratedincreased liver uptake but lower kidney and bladder activities comparedto the control group. Interestingly, although the heart displayed verylittle retention of this molecular imaging agent, the uptake as measuredex vivo was increased in the PAH animals from 0.18±0.03% ID to0.77±0.46% ID, P=0.02. The increased uptake in the right heart ventriclecorrelated with RV weight (r=0.83, P<0.01) while there was nocorrelation for the left ventricle+septum (r=−0.58).

Whole body images of rats 30 minutes after injection are presented inFIG. 28. There is homogeneous bilateral lung uptake in control rats withmarked reduction of ^(99m)Tc-AM-L in the animals with PAH as there isbarely any visibly evident lung uptake.

The heterodimeric AM receptor component, receptor activity modifyingprotein 2 (RAMP2), was evaluated in lung tissue by western immunoblots.There was marked reduction of RAMP2 protein expression (FIG. 29) in PAHrats (P<0.001).

Discussion

A linear human AM derivative radiolabelled with ^(99m)Tc was used forimaging of the pulmonary circulation and tested its ability to diagnosePAH. It was demonstrated that a molecular imaging agent can be used todetect abnormalities of the pulmonary microcirculation. In PAH, lunguptake of ^(99m)Tc-AM-L was markedly reduced.

PAH is a disorder characterized by medial hypertrophy of pulmonaryarterioles with intimal proliferation leading to obliteration and lossof pulmonary circulation. There currently exists no test that cannon-invasively detect this loss of pulmonary micro-circulation. The MCTmodel of PAH, although lacking the intimal proliferation of human PAH,is similarly associated with medial hypertrophy with obliteration andloss of pulmonary arterioles [41, 42]. The observed reduction in^(99m)Tc-AM-L uptake in PAH could therefore in great part be caused byreduced pulmonary vascular surface with loss of AM receptors. However,other mechanisms could be involved without limiting the scope of thepresent invention.

The AM receptor is a heterodimeric G-protein coupled receptor composedof two components, the calcitonin receptor like receptor (CLR) and areceptor activity modifying protein (RAMP2) [43]. Large-scale analysisof the human and mouse transcriptomes revealed that RAMP2 is relativelyequally distributed among most tissues, with the notable exception ofvery high expression levels in the lungs (44). Human and rat lungsindeed contain a high density of specific AM binding sites (39, 40)mostly distributed on the vascular endothelium. This is concordant withstudies demonstrating that the lung is an important site for circulatingAM clearance [37, 38]. Acute lung injury in a sepsis model is associatedwith markedly increased circulating AM levels with concomitant 95%reduction in lung RAMP2 expression, suggesting that reduced lung bindingand clearance could contribute to the increased plasma levels [45]. Inthe current study, we also evaluated lung RAMP2 protein expression andfound that it was markedly reduced by about 80%. This is consistent withthe approximate 70% reduction in lung uptake that we found and with theincreased (doubling) of plasma 99mTc-AM-L levels in PAH.

An interesting and unexpected finding was the increase in the heartuptake of ^(99m)Tc-AM-L in PAH that correlated with the severity ofright ventricular hypertrophy. Although the expression of AM receptorswas not evaluated in the RV, this would suggest that AM receptors arepresent and that their expression is increased by RVH. Whether increaseduptake by the RV could be detected clinically and serve as an index ofRVH would require further validation but this would certainly provideuseful additional information.

The MCT model of lung injury with PAH is not selective to the pulmonarycirculation. Another organ sensitive to the effect of MCT is the kidneyand previous studies have used MCT injection as a model of renal injury[46]. Although this study was not specifically designed to evaluatekidney function, we found that MCT resulted in reduced kidney uptake of^(99m)Tc-AM-L by about 50%. This suggests that loss of kidney AM uptakecould be used to evaluate kidney damage in this model but furtherstudies specifically evaluating the kidneys AM system in this model andothers.

EXAMPLE 13 Additional Derivatives and their Clinical Properties

FIG. 30 illustrates in table form and summarizes some of the salientfeatures of additional experiments with various AM derivatives. In thistable, the “base” AM is hAM as identified in SEQ ID NO: 01. Thesequences of these derivatives are depicted by comparison to humancyclic AM (the first listed). Through novel modifications in thepeptides, we have created new compounds that have potential clinicaladvantages. Various AM derivatives that were investigated for differentproperties. These derivatives were synthesized as follows.

All adrenomedullin (AM) analogs were synthesized through solid phasepeptide synthesis following a standard procedure for Fmoc chemistry. ARink AM resin was chosen as the solid support. Amino acids wereincorporated to the peptide chain in accordance with the AM(21-52)sequence of the natural peptide. In cases in which PEG subunits areinserted, a Fmoc-dPEG₂-OH or Fmoc-dPEG₄-OH derivative was added toreplace amino acids found between cysteine residues of the complete AMpeptide while maintaining dimensions similar to those of the nativepeptide. The coupling protocol used for all amino acids was alsofollowed for the PEG including AM derivatives. A cysteine residue wascoupled onto the deprotected amine group of the PEG spacer and finally,the chelating moiety, if present and which corresponds in some examplesto a 4-amino acid sequence, was attached to the peptide chain followingthe same peptide synthesis procedure. More details concerning methodsusable for synthesizing such AM derivatives are found in previousexamples.

By removing the 1-12 fragment of hAM, we demonstrate that the fragment1-12 is not essential either for binding to the lung or for hemodynamicactivity. Derivatives that have 13-52 morphology are sufficient. This isseen by the data concerning the biodistribution of AM derivativesincluding and excluding these fragments in dogs after 30 min accordingto the protocols described hereinabove and illustrated in FIG. 31. Theseexperiments establish that the presence of the two cysteine residues isimportant also for receptor binding (lung imaging) and for activity ofthe derivatives.

It was also established that by introducing a spacer between the twocysteine residues (polyethylene glycol, PEG2 and PEG4), good lungbinding and imaging was obtained, but reduced unwanted hypotensiveeffects. Furthermore, replacement of the amino-acids in position 13, 14and 15 by the chelator Gly-Gly-dAla-Gly (GGAG) results in enhancedlabelling of the tracer with Tc99M.

Furthermore, we demonstrate that the cyclic derivative with a spacer(PEG4) and the chelator GGAG provides the best lung kinetics using aprotocol substantially similar to the protocols described hereinabove,as seen in FIG. 33, with a plateau effect as the tracer is retained in amore stable fashion by the lungs over 1 hour.

Although the present invention has been described hereinabove by way ofpreferred embodiments thereof, it can be modified, without departingfrom the spirit and nature of the subject invention as defined in theappended claims.

All references cited and/or discussed in this specification areincorporated herein by reference in their entirety and to the sameextent as if each reference was individually incorporated by reference.

The in vivo experiments in rats and dogs, as described in thespecification, may be predictive of biological effects in humans orother mammals and/or may serve as animal models for use of the presentinvention in humans or other mammals.

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1. An adrenomedullin derivative comprising: an adrenomedullin peptide ora fragment thereof bound with at least one active agent, theadrenomedullin peptide being a mammalian adrenomedullin peptide.
 2. Theadrenomedullin derivative of claim 1, wherein the active agent comprisesa radioactive element.
 3. The adrenomedullin derivative of claim 2,wherein the adrenomedullin peptide comprises a peptide having thesequence:Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr(SEQ ID NO: 1) or a fragment thereof.
 4. The adrenomedullin derivativeof claim 3, wherein the adrenomedullin peptide is in a linear form. 5.The adrenomedullin derivative of claim 3, wherein the adrenomedullinpeptide is in a cyclic form.
 6. The adrenomedullin derivative of claim3, wherein the adrenomedullin peptide comprises an adrenomedullinfragment having the sequence:Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr(SEQ ID NO: 2) or a fragment thereof.
 7. The adrenomedullin derivativeof claim 3, wherein the adrenomedullin peptide comprises anadrenomedullin fragment having the sequence:X1-X2-X3-X4-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr(SEQ ID NO:3), wherein: X1 is absent or is selected from the groupconsisting of:Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly, Ser-Phe-Glyand Gly-Gly-Ala-Gly; X2 is Cys or HomoCys; X3 is Arg-Phe-Gly-Thr or alinear or branched PEG moiety having at least two Poly(ethylene glycol)(PEG) subunits; and X4 is Cys or HomoCys.
 8. The adrenomedullinderivative of claim 7, wherein X3 is a linear PEG moiety having 2 or 4PEG subunits.
 9. The adrenomedullin derivative of claim 7, wherein X1 isGly-Gly-dAla-Gly.
 10. The adrenomedullin derivative of claim 7, whereinX1 is Gly-Gly-Ala-Gly, X2 is Cys, X3 is a linear moiety having 2 or 4PEG subunits, and X4 is Cys.
 11. The adrenomedullin derivative of claim7, wherein the adrenomedullin peptide is in a cyclic form.
 12. Theadrenomedullin derivative of claim 3, wherein the radioactive element isbound directly to an amino acid of the adrenomedullin peptide.
 13. Theadrenomedullin derivative of claim 12, wherein the radioactive elementis ^(99m)Tc.
 14. The adrenomedullin derivative of claim 12, wherein theradioactive element is selected from the group consisting of: ^(99m)Tc,⁶⁷Ga, ⁶⁴Cu, ⁹⁰Y, ¹⁶¹Tb, ¹⁷⁷Lu, and ¹¹¹In.
 15. The adrenomedullinderivative of claim 12, wherein the adrenomedullin peptide is in alinear form.
 16. The adrenomedullin derivative of claim 12, wherein theradioactive element is bound directly to a cysteine amino acid of theadrenomedullin peptide.
 17. The adrenomedullin derivative of claim 1,wherein the active agent comprises a paramagnetic element.
 18. Theadrenomedullin derivative of claim 1, wherein the active agent is anelement selected from the group consisting of: Fe, Ca, Mn, Mg, Cu, andZn.
 19. The adrenomedullin derivative of claim 1, wherein the activeagent is selected from the group consisting of: active agents comprisingat least one paramagnetic element, active agents comprising at least oneradioactive element, and fibrinolytic agents.
 20. A method ofdetermining a disease state in an organ in a mammal, the organcomprising adrenomedullin-receptor-bearing cells, the method comprising:a) administering to the mammal a labelled adrenomedullin derivative inan effective amount to achieve binding between the labelledadrenomedullin derivative and the adrenomedullin-receptor-bearing cells;b) generating an image of the distribution of the labelledadrenomedullin derivative in the organ of the mammal; c) using the imageof step b) to determine a labelling pattern of the adrenomedullinderivative in the organ; d) comparing the labelling pattern of step c)to a labelling pattern of a non diseased organ; and e) determining thedisease state of the organ at least in part on the comparison of stepd).
 21. The method of claim 20 wherein the organ is a lung.
 22. Themethod of claim 21, wherein the disease is pulmonary embolus and whereinthe labelling pattern of step c) indicates that the labelledadrenomedullin derivative is present in a greater concentration inupstream regions of the lung than in downstream regions of the lung. 23.The method of claim 22, wherein detecting the pulmonary embolus includesidentifying a labelling pattern in a region of the lung in which thelabelled adrenomedullin derivative is present in a lower concentrationthan in adjacent regions of the lung.
 24. The method of claim 21,wherein the disease is pulmonary arterial hypertension.
 25. The methodof claim 24, wherein determining the presence of pulmonary arterialhypertension includes detecting a reduced uptake of the labelledadrenomedullin derivative as compared to a baseline uptake of thelabelled adrenomedullin derivative in the non diseased organ, whereinthe reduced uptake indicates pulmonary arterial hypertension.
 26. Themethod of claim 20, wherein the organ is a kidney.
 27. The method ofclaim 26, wherein the disease is kidney damage.
 28. The method of claim27, wherein determining the kidney damage includes identifying alabelling pattern in a region of the kidney in which the labelledadrenomedullin derivative is present in a lower concentration than inadjacent regions of the kidney.
 29. The method of claim 27, whereindetermining the kidney damage includes detecting a reduced uptake of thelabelled adrenomedullin derivative as compared to a baseline uptake ofthe labelled adrenomedullin derivative in the non diseased organ,wherein the reduced uptake indicates kidney damage.
 30. The method ofclaim 20, wherein the mammal is human.
 31. The method of claim 20wherein the labelled adrenomedulin derivative is administered to themammal at a substantially hemodynamically inactive dose.
 32. The methodof claim 20, wherein the labelled adrenomedullin derivative is labelledwith a radioactive element.
 33. The method of claim 20, wherein thelabelled adrenomedullin derivative comprises: an adrenomedullin peptideor a fragment thereof bound with a radioactive element, theadrenomedullin peptide being a mammalian adrenomedullin peptide.
 34. Themethod of claim 33, wherein the adrenomedullin peptide comprises anadrenomedullin fragment having the sequence:X1-X2-X3-X4-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr(SEQ ID NO:3), wherein: X1 is absent or is selected from the groupconsisting of:Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly, Ser-Phe-Glyand Gly-Gly-Ala-Gly; X2 is Cys or HomoCys; X3 is Arg-Phe-Gly-Thr or alinear or branched PEG moiety having at least two Poly(ethylene glycol)(PEG) subunits; and X4 is Cys or HomoCys.
 35. The method of claim 34,wherein X3 is a linear PEG moiety having 2 or 4 PEG subunits.
 36. Themethod of claim 34, wherein X1 is Gly-Gly-dAla-Gly.
 37. The method ofclaim 34, wherein X1 is Gly-Gly-Ala-Gly, X2 is Cys, X3 is a linearmoiety having 2 or 4 PEG subunits, and X4 is Cys.
 38. The method ofclaim 34, wherein the adrenomedullin peptide is in a cyclic form. 39.The method of claim 34, wherein the radioactive element is bounddirectly to an amino acid of the adrenomedullin peptide.
 40. The methodof claim 34, wherein the radioactive element is ^(99m)Tc.
 41. The methodof claim 34, wherein the adrenomedullin peptide is in a linear form. 42.The method of claim 34, wherein the radioactive element is bounddirectly to a cysteine amino acid of the adrenomedullin peptide.
 43. Themethod of claim 32, wherein the labelled adrenomedulin derivative isadministered to the mammal by injection comprising from about 0.5 mCi toabout 1000 mCi of labelled adrenomedulin derivative.
 44. The method ofclaim 20, wherein the labelled adrenomedullin derivative is dissolvedinto a buffer solution, administering the labelled adrenomedullinderivative to the mammal comprising injecting into the bloodstream ofthe mammal the labelled adrenomedullin derivative dissolved into thebuffer solution.
 45. A method of determining the presence and density ofadrenomedullin receptor-bearing cells in a mammal comprising:administering to the mammal an effective amount of labelledadrenomedullin derivative to achieve binding between the labelledadrenomedullin derivative and adrenomedullin-receptor-bearing cells; anddetermining the distribution of the labelled adrenomedullin derivativeto obtain an image of the adrenomedullin-receptor-bearing cells.
 46. Themethod of claim 45, wherein the labelled adrenomedullin derivativecomprises: an adrenomedullin peptide or a fragment thereof bound with aradioactive element, the adrenomedullin peptide being a mammalianadrenomedullin peptide.
 47. The method of claim 46, wherein theadrenomedullin peptide comprises an adrenomedullin fragment having thesequence:X1-X2-X3-X4-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr(SEQ ID NO:3), wherein: X1 is absent or is selected from the groupconsisting of:Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly, Ser-Phe-Glyand Gly-Gly-Ala-Gly; X2 is Cys or HomoCys; X3 is Arg-Phe-Gly-Thr or alinear or branched PEG moiety having at least two Poly(ethylene glycol)(PEG) subunits; and X4 is Cys or HomoCys.
 48. An adrenomedullinderivative comprising: an adrenomedullin peptide, the adrenomedullinpeptide comprising a peptide having the sequence:Tyr-Arg-Gln-Ser-Met-Asn-Asn-Phe-Gln-Gly-Leu-Arg-Ser-Phe-Gly-Cys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr(SEQ ID NO:1) or a fragment thereof, the adrenomedullin peptide being amammalian adrenomedullin peptide.
 49. The adrenomedullin derivative ofclaim 48, wherein the adrenomedullin peptide is in a linear form. 50.The adrenomedullin derivative of claim 48, wherein the adrenomedullinpeptide comprises an adrenomedullin fragment having the sequence:X1-X2-X3-X4-Thr-Val-Gln-Lys-Leu-Ala-His-Gln-Ile-Tyr-Gln-Phe-Thr-Asp-Lys-Asp-Lys-Asp-Asn-Val-Ala-Pro-Arg-Ser-Lys-Ile-Ser-Pro-Gln-Gly-Tyr(SEQ ID NO:3), wherein: X1 is absent or is selected from the groupconsisting of: Ser-Phe-Gly and Gly-Gly-Ala-Gly; X2 is Cys or HomoCys; X3is Arg-Phe-Gly-Thr or a linear or branched PEG moiety having at leasttwo Poly(ethylene glycol) (PEG) subunits; and X4 is Cys or HomoCys. 51.The adrenomedullin derivative of claim 50, wherein X3 is a linear PEGmoiety having 2 or 4 PEG subunits.
 52. The adrenomedullin derivative ofclaim 50, wherein X1 is Gly-Gly-Ala-Gly, X2 is Cys, X3 is a linearmoiety having 2 or 4 PEG subunits, and X4 is Cys.
 53. The adrenomedullinderivative of claim 50, further comprising a radioactive element bounddirectly to an amino acid of the adrenomedullin peptide.
 54. Theadrenomedullin derivative of claim 53, wherein the radioactive elementis ^(99m)Tc.
 55. The adrenomedullin derivative of claim 54, wherein theadrenomedullin peptide is in a linear form.
 56. The adrenomedullinderivative of claim 54, wherein the radioactive element is bounddirectly to a cysteine amino acid of the adrenomedullin peptide.
 57. Theadrenomedullin derivative of claim 50, wherein the adrenomedullinpeptide is bound to at least one active agent.
 58. The adrenomedullinderivative of claim 50, further comprising a chelating peptidecovalently bound to the adrenomedullin peptide and at least one activeagent bound to the chelating peptide.
 59. The adrenomedullin derivativeof claim 58, wherein the active agent comprises a radioactive element.